Local, site-specific characteristics largely control the transmission dynamics of arthropod-borne viruses (arboviruses). Arboviruses, in turn, adapt to local conditions, maximizing their potential to perpetuate and emerge as health threats. The adaptive potential of arboviruses is driven by error-prone replication, which creates a genetically diverse pool of competing virus genotypes within each host. This proposal examines how mosquitoes and birds act in concert to shape WNV evolution and fitness. Our previous research has allowed us to make very clear predictions about the outcome of each proposed aim and has facilitated our ability to translate our previous work to new emerging pathogens such as Zika virus. In birds, WNV fitness gains are limited by high MOI environments in susceptible vertebrates (e.g. crows) and promoted in birds that limit replication (e.g. robins). Mosquitoes also have species-dependent impacts on WNV diversification and fitness. Ironically, systemic infection of mosquitoes leads to reduced fitness in transmitted WNV populations. Therefore, Aim 1 will attempt to either reduce or increase WNV fitness by forcing it into transmission cycles with different host assemblages. We predict that crows and Cx. pipiens mosquitoes will result in WNV populations that are dramatically reduced in fitness compared to WNV that is maintained by robins and Cx. quinquefasciatus. Our results strongly suggest that the limitations on fitness gains of WNV when it replicates in crows are related to the high viremias that occur in this host relative to robins. At high MOI, coinfection of individual cells is efficient and defective (or low fitness) genomes are complemented by those of high fitness. This suppresses the overall fitness of the population. Aim 2 of the current proposal tests this hypothesis through in vivo and ex vivo studies of WNV loads and diversity in avian PBMCs, a critical site of WNV replication. We predict that at high MOI, clearly deleterious mutations (intrahost length-variants, for example) will persist and fitness will be reduced. The fitness declines that we observed in WNV during mosquito infection occur because of high virus mutation rates coupled with stochastic reductions in the population (i.e. bottlenecks) as the virus moves from one mosquito tissue to another. It is therefore critical to understand the mechanistic basis for the formation of these ?barriers? to arbovirus transmission. Our preliminary data suggests that one critical aspect that contributes to them is RNAi-based targeting of the flavivirus sfRNA. In addition, we have preliminary data suggesting that sfRNA1/2 facilitates virus escape from anatomical barriers. Therefore, in Aim 3 we will examine how mosquito RNAi targets the WNV genome, and in particular the sfRNA1 start site, and how the virus population changes as a result of being ?trapped? within a transmission barrier. This aim also will leverage our extensive experience working on WNV-host interactions to more deeply understand the emergent Zika virus. To accomplish this we will use newly developed reverse genetics systems for WNV and ZIKV that lack the ability to produce sfRNA1. Preliminary data on this is provided in the application. The significance of this work is that it will provide novel data on how different transmission cycles can impact virus genetics, and how this can lead to the emergence of new virus strains. Our proposed work will also provide important mechanistic data on why different birds and mosquitoes have different impacts on virus populations. Translating our WNV-based findings to ZIKV is also critical to this work, as we think it is our job to use what we have learned to address new arboviral threats. Finally, the significance of our work is that we have provided technical and analytical tools that are broadly useful and have permitted us to collaborate effectively with a wide array of investigators. The proposed studies are technically and conceptually innovative due to, for example, our modeling of the WNV transmission cycle, our use of single-cell approaches, and our ability to profile small RNAs within mosquito salivary glands.